The expression of the aryl hydrocarbon nuclear translocator (ARNT and ARNT2) proteins are essential for development of multiple organ systems in mammals and are required for physiological pathways that respond to important exogenous and endogenous environmental signals (chemicals, hormones, oxygen). Reductions in ARNT protein expression in the pancreas have been implicated in type 2 diabetes, while elevated expression of its bHLH/PAS dimerization partner SIM2, has been implicated in Downs Syndrome. ARNT and ARNT2 proteins exhibit 80-90% amino acid identity in the bHLH and PAS domains. There is conflicting data in the literature regarding the potential for these two proteins to perform the same function in vitro and other conflicting data concerning the pattern of expression of ARNT2 in vivo and in cell lines. Given the importance of these proteins in many of the same signaling pathways, it is intriguing that targeted disruption of the Arnt gene in mice results in embryonic lethality due to vascular defects, while the targeted disruption of the Arnt2 gene is characterized by perinatal lethality involving CNS deficits. Despite the overlaps in tissue expression and the high level of amino acid identity between these two proteins, the prevailing interpretation of the transgenic results has resulted in the hypothesis that ARNT and ARNT2 proteins are not capable of compensating for the loss of the other protein in vivo. However, there are several explanations that may account for these findings such as i) each ARNT has a distinct level of protein expression in a given tissue that does not support a specific pathway, ii) tissue specific modifications of the different ARNTs impact their function, or iii) each ARNT has one or more distinct functions not shared by the other. A powerful approach to begin to address these important questions is to utilize a knockin/knockout strategy whereby the ARNT2 ORF is inserted into the Arnt locus while simultaneously abolishing ARNT protein expression. In this approach, ARNT2 expression will be controlled by all the regulatory elements normally reserved for ARNT and result in the physiological expression of ARNT2 in a mouse that does not express ARNT. The generation of this novel gene-targeted model will allow a direct test of the hypothesis that the concentration of ARNT2 protein expression in tissues contribute to the lack of compensation in Arnt-/- animals. The integrated set of aims will show how the level of expression of both ARNT and ARNT2 protein impact established physiological pathways, the response to important exogenous and endogenous environmental signals and development. Successful completion will illustrate the utility of such a transgenic approach to assess the function of similar proteins, but also may produce key models for the analysis of development (via SIM and HIF proteins), pharmacology and toxicology (via HIF and AHR proteins), diabetes (via reductions in ARNT) and cancer (via AHR and HIF proteins). PUBLIC HEALTH RELEVANCE: The expression of the aryl hydrocarbon nuclear translocator (ARNT and ARNT2) proteins are essential for development of multiple organ systems in mouse and are required for established physiological pathways that respond to important exogenous and endogenous environmental signals (chemicals, hormones, oxygen). ARNT is a key protein in development of cancer and perturbation of ARNT and ARNT2 expression has been associated with diabetes and neurological disorders.